The dynamic interactions of the intestinal epithelium with the intestinal microbiota require the co-ordination of the barrier function with host defense responses to maintain intestinal homeostasis. However, the mechanisms involved in this eukaryotic and prokaryotic cross-talk are only partially understood. To overcome the intestinal barrier, several enteric pathogens have evolved distinct strategies to interfere with the function of tight junctions, a specialized membrane domain at the most apical region of intestinal epithelial cells that maintains the cellular polarity and creates a selective and regulated barrier for ions and macromolecules. The outlined experiments will continue our goal to unravel the mechanisms, which facilitate the maintenance of the intestinal barrier function during host defense responses. During the previous funding period, we have identified a guanine nucleotide exchange factor for RhoA, GEF-H1, in tight junctions of intestinal epithelial cells. GEF-H1 may be central to epithelial cell defenses to pathogens by controlling the formation of tight junctions, cell invasion by pathogens, and activation of inflammatory host responses. The mechanisms, which localize GEF-H1 in tight junctions, and those responsible for its release and initiation of signaling events in intestinal epithelial cells are unknown. We hypothesize that GEF-H1 is a key regulator of Rho GTPase dependent mechanisms in tight junctions; which together, determine the adaptation of the intestinal barrier function to challenges by commensal and pathogenic bacteria. The overall goals of this proposal are to characterize the biological functions of GEF-H1 in the regulation of the intestinal epithelial barrier function and to define the molecular mechanisms, which are responsible for GEF-H1 mediated protection from pathogen cell invasion.